Abstract 2028: Protein O-GlcNAcylation modulates the secretome of keratinocyte and its paracrine regulation of fibroblast function

Abstract


Parisa Raeisimakiani, Lara Mahal, Rebecca Powell
Breast milk is generally accepted as the perfect source of nutrition for the health and development of infants. It also assists in infant innate and adaptive immunity through many proteins that are decorated with glycans. Examples of these glycoproteins include IgA, IgG, and innate immune lectins. Maternal diet and environmental exposure such as pathogens and pollutants affect human milk composition including its glycoprofile. Despite altered glycosylation can have a consequence on the nursing infant's health and immunity, the current knowledge is still emerging in this area of study. COVID-19 has gained attention in recent years by causing severe morbidity and mortality. Similar to other infectious diseases such as influenza, our lab recently revealed alterations in glycome of plasma and different tissue samples of COVID-19 infected patients. Inspired by these findings, we are interested in disclosing the effect of SARS-CoV-2 on glycosylation of breast milk proteins. Toward this, we performed a large-scale systematic study using our high-throughput lectin microarray analysis technology. We analyzed 132 control samples (breast milk collected pre-COVID) and breast milk from 78 COVID-19 infected mothers. Our data showed there is a 4-fold increase in ∝-2,3 sialic acid on glycoproteins that is associated with SARS-CoV-2 infection in lactating mothers. Lectin pulldown experiments further testified to these findings. Given the significance of ∝-2,3 sialic acid glycan signature in infectious diseases, our finding could provide valuable insight into therapeutic development. Background: Keratinocytes in the epidermis of skin have a crucial role in regulating the functions of fibroblasts in dermis by secreting pro-inflammatory and pro-fibrotic cytokines via paracrine effects. Abnormal changes of this paracrine effect may contribute to the dysregulated fibrosis in diseases such as scleroderma. Protein O-GlcNAcylation is the addition of the GlcNAc moiety from nucleotide uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) onto serine or threonine residue of cytosolic proteins. This is catalyzed by O-GlcNAc transferase (OGT), and removed by O-GlcNAcase (OGA). Abnormal levels of O-GlcNAcylation of proteins are found in various chronic diseases including diabetes, cardiovascular diseases, cancers and Alzheimer's disease. Objective of this study was to determine the role of protein O-GlcNAcylation in regulating secretion of cytokines by keratinocytes and the consequent impact on fibroblasts function. Methods Both primary keratinocytes and dermal fibroblasts were isolated from the wildtype C57BL/6 mouse and cultured separately in vitro. The keratinocytes were treated with a chemical inhibitor to either OGT (OSMI-1) or OGA (Thiamet-G) for 24 hours. At the end of treatment, the inhibitor-containing media was removed and keratinocytes were cultured in fresh media for 48 hours. The keratinocyte-conditioned media (K-CM) was then transferred from the keratinocytes to fibroblasts. After 48-hour culture in K-CM, the fibroblasts were harvested for further analysis of markers for fibrosis. Alternatively, the K-CM was processed for secretome analysis by Mass Spectrometry. The inhibitor-treated donor keratinocytes were harvested for analysis of markers for terminal differentiation, apoptosis, autophagy, and cell senescence. In addition, transcriptomic analyses of the donor keratinocytes by RNA Sequencing were carried out to explore alterations in global transcription. Results 1), Chemical inhibition of OGT significantly impaired the terminal differentiation of keratinocytes, which was conversely enhanced by chemical inhibition of OGA. However, neither treatment had any effect on apoptosis or autophagy of keratinocytes; 2), Transcriptomic analyses by RNA Seq revealed global transcription changes in various signaling pathways; 3), Secretome analysis of K-CM revealed that the levels of some profibrotic cytokines including Connective Tissue Growth Factor and Fibroblast Growth Factor 21, etc. were significantly lower, while the levels of some anti-inflammatory proteins such as Heme Oxygenase-1 were significantly higher, in OSMI-1-treated K-CM; 4), The gene expressions of both Acta2 and Collagen I were significantly downregulated in fibroblasts cultured in OSMI-1treated K-CM. 5), Culture in OSMI-treated K-CM also induced significant caspase-dependent apoptosis in fibroblasts.
Conclusions These findings demonstrate that inhibition of protein O-GlcNAcylation in keratinocytes alters their production of certain cytokines that regulate activation and turnover of fibroblasts. Protein O-GlcNAcylation may be a novel therapeutic target in treating dysregulated fibrosis in diseases such as scleroderma. We are currently combining secretome analysis with an antibody-based multiplex cytokine assay to identify candidate cytokines that are regulated by protein O-GlcNAcylation in keratinocytes. In addition, we are validating the cell culture findings in an in vivo mouse model of skin fibrosis.
This study was supported by NIH1K12HL14195 (to Vincent C. Hascall) and the National Scleroderma Foundation New Investigator Award (to Yan Wang). O-GlcNAcase (OGA), which catalyze the addition and removal of UDP-GlcNAc on the protein, respectively. O-GlcNAcylation utilizes glucose for UDP-GlcNAc synthesis. Glucose metabolism is complex, being used for energy production as well as for biomolecule synthesis. The level of protein O-GlcNAcylation in asthma has not been described although energy metabolism is altered in asthma and linked to asthma pathophysiology. Here, we investigate protein O-GlcNAcylation in asthma and the impact of glucose energy metabolism on protein O-GlcNAcylation. We hypothesize that glucose metabolism regulates protein O-GlcNAcylation, and inhibition of glucose oxidation will increase glucose availability for UDP-GlcNAc synthesis, leading to overall increased protein O-GlcNAcylation.
Methods: To test this hypothesis, human airway smooth muscle cells (HASMCs) were isolated from lungs of asthmatics (n = 5) and non-asthmatic controls (n = 5). Cells were assessed at baseline culture and with blockade of mitochondrial glucose oxidation by UK5099, an inhibitor of mitochondria pyruvate carrier 1. Protein O-GlcNAcylation (detected by CTD110.6 antibody), OGT, OGA, and AMP-activated protein kinase proteins (Western Blotting) and cellular bioenergetics (Seahorse) were evaluated at baseline and in response to UK5099 in non-asthmatic and asthmatic HASMCs.
Results: OGT and OGA proteins were similar between asthmatic and non-asthmatic cells at baseline. Despite similar level of protein O-GlcNAcylation, non-asthmatic and asthmatic HASMCs had distinct differences in protein O-GlcNAcylation at baseline. Inhibition of mitochondrial glucose oxidative metabolism in HASMCs with UK5099 (10 µM) increased the protein O-GlcNAcylation in non-asthmatic control cells (1.6 ± 0.5-fold, p < 0.05), but not in asthmatic cells (0.9 ± 0.2fold, p = 0.34). OGT and OGA proteins were not affected in response to UK5099 treatment in either non-asthmatic or asthmatic HASMCs. In asthmatic, but not in non-asthmatic cells, UK5099 decreased mitochondrial respiration by 38 ± 5% ( p < 0.01), increased glycolytic reserve capacity by 40 ± 15% ( p = 0.06), and increased phosphorylation of the intracellular nutrient and energy sensor AMP-activated protein kinase at threonine 172 (1.5 ± 0.1-fold, p < 0.01). The greater AMPK phosphorylation occurs upon cell sensing lower energy status, and stimulates pathways for energy (ATP) production while suppressing pathways that utilize ATP.